Of all the domestic and cultivatable plants, cotton is one of the most attacked by diseases and insect-pests, besides being highly sensitive to the occurrence imposed by weed plants (Beltrão, E. M., Souza, J. G. O. agronegócio do algodão no Brasil. Embrapa: Brasília, v. 01, 1999). Among the main insect-pests comes the boll weevil, Anthonomus grandis (Boheman, C. H. Description of new species. In Schoenherr, Genera et species Curculionidum cum synonymia hujus Familiae, vol. 7, pt. 2. Paris: Roret. 461 p., 1843), considered one of the most serious pests for cotton cultivation, being prevalent across Mexico, Cuba, Haiti, Venezuela, Colombia, Paraguay and Brazil. This insect uses the flower buds and fruits of its host as a source of food and place for development, causing direct prejudice to the commercialization of cotton fiber. Levels of infestation grow rapidly and up to 100% of production can be affected unless adequate control measures are taken. This insect represents potentially major damage, being considered a key pest in the planning and control of insects that are harmful to crops, mainly due to the difficulty of control by chemical insecticides.
The cotton plant and its pests have co-existed for a long evolutionary period.
Plant and insect form an interdependent and competitive morphological and biochemical system, most often resulting in the use of part of the plant by the insect. This part used represents the damage caused by the insect to the plant, and depends on the size of the pest population, and the plant's ability to resist the attack and to recover from the damage sustained (Beltrão, E. M., Souza, J. G. O. agronegócio do algodão no Brasil. Embrapa: Brasília, v. 01, 1999).
The plant versus insect interaction can be visualized in at least two ways: from the point of view of the insect, with the plant varying from suitable to completely unsuitable as host and, on the other hand, from the point of view of the plant where, the lower the number of species and abundance of insects associated thereto, and the lesser the effect that these insects exert thereover, the greater their resistance to these insects (Santos, W. J. Identificação, biologia, amostragem e controle das pragas do algodoeiro. In: Embrapa Agropecuária Oeste; Embrapa Algodão. Algodão: tecnologia de produção., p. 296 p. 2002).
Between one extreme and another of plant resistance to insect-pests, there is a complete and extensive arsenal of mechanisms to attack and counter-attack the action of insects, which include from a simple morphological impediment to complex phytochemical components, which interfere directly in the metabolic process involved in the use of the plant as insect host. In practical terms, the resistance of the cotton plant to insect-pests represents the ability of certain crops to produce better quality cotton in a greater amount than other crops, under attack from the same population of insect-pests (Freire, E. C. Cultivares e produção de semente na melhoria da qualidade do algodão no nordeste e centro-oeste do Brasil. Boletim informativo Embrapa/CNPA. 1997).
In most countries where cotton is cultivated, vulnerability to pests represents the main problem of this crop. Having no alternatives for more effective control, the producers routinely continue to believe that chemical insecticides are the only way to protect the crops. Though efficient, they are expensive, potentially harmful to man, to the environment and, in the long term, trigger resistance processes, pest's resurgence and reduction in the incidence of natural enemies (Panizzi, A. R. Efeito de insecticides na população das principais pragas da soja. An. Soc. Entomol. Brasil, v. 6, p. 264-275. 1977). Under these circumstances, the objective of the present invention is to increase the resistance of plants, generating transgenic plants, which are capable of expressing genes with high entomotoxic activity, whereby solving the problem of the abusive use of chemical insecticides.
The stable introduction of exogenous genes into cotton plants, with the purpose of inducing resistance to insect-pests, is an excellent alternative to reduce a large part of the problems associated to chemical methods. This technology comprises various advantages, chiefly because it does not pollute the environment. General data demonstrate that transformed cotton plants does not present negative effects to the environment, the characteristics being inheritable and expressed normally in the plant (Adamczyk, J. J., L. C. Adams L. C., Hardee, D. D. Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes. Journal of Economic Entomology, v. 94, n.6, DEC, p. 1589-1593. 2001).
The availability of microorganisms and organic compounds in nature for biological use is very widespread, and they supply a wide variety of raw materials for the development of new products, having greater pathogenicity against the insect and broad action spectrum. Among these micro-organisms, a major discovery was the soil bacteria Bacillus thuringiensis, which is widely used as a biological control agent and as a source of potential molecules for biotechnological programs, destined to obtain transgenic plants resistant to insect-pests. With this strategy, it is possible to reduce populations of agricultural pests of economic interest to tolerable levels (Perlak, F. J., R. W. Deaton, T. A. Armstrong, R. L. Fuchs, S. R. Sims, J. T. Greenplate and D. A. Fischhoff. Insect resistant cotton plants. Biotechnology (NY), v. 8, n. 10, p. 939-943. 1990).
Although some δ-endotoxins with activity on the boll weevil have already been identified and described, the endophytic habit of this pest hampers or even prevents the use of these toxins by conventional means, which are commercialized as bioinsecticides, such as, for example, protein formulations containing Cry toxins. They present instability in the environment, low yield in purification from natural sources, in addition to easy loss of the activity of these toxins by weather conditions such as rain and sun. Faced with this problem, the most efficient strategy is the use of Cry toxin-encoding genes in the generation of genetically-modified plants.
The use of encoding genes for this type of entomotoxic proteins and the expression of same in heterologous systems (bacteria or transgenic plants) overcomes the difficulties caused by the use of bioinsecticides. This strategy has gained prominence in recent years in the field of transgenia, due to the specificity of these toxins in relation to the insect-pests, efficiency, driven expression and innocuity to animals and humans. Accordingly, genetically-modified plants with specific resistance to insect-pest can be generated in high efficiency systems.
There are some Bt genes and transgenes with activity for coleoptera, such as, for example, the plants expressing a cry8 gene by the company DU PONT DE MENOURS with toxicity for Leptinotarsa decemlineata (US20030177528), the transgenic corn with a cry8-like gene by PIONEER & DU PONT with toxicity for Diabrotica virgifera, Diabrotica undecimpunctata howardi, Leptinotarsa decemlineata and Anthonomus grandis (US20060021096, as also mentioned in U.S. Pat. No. 7,105,332 and US2005166284), Feng, S et al., 2005 also describe a modified cry8 gene, cry8Ca, with specific activity for coleoptera insects (CN1609220-A) and, more recently, PIONEER & DU PONT describes a synthetic cry8 gene with toxicity for Diabrotica virgifera virgifera in monocot plants such as, for example, corn plants (as mentioned in patent application US20060288448).
Currently, plants expressing genes Bt of the cry8 type are, in their totality, monocot (eg.: corn). This being the case, to-date, no invention has described a gene of this nature, with potential application in dicot plants, as is the case of the cotton plant.
Modern techniques of molecular biology, such as the construction of combinatorial libraries, are used to develop and identify analog mutant genes with specific objectives.
Construction of variant analog genes libraries using molecular evolution technology in vitro, have been used over the last three decades. This fact is due to the appearance of biotechnological tools, which act as a platform for genetic engineering in the development of new molecules with improved activity, mainly intended for agriculture and the pharmaceuticals industry (Ling Yuan, L. Kurek, I., English, J. and Keenan, R. Laboratory-directed protein evolution. Microbiology and Molecular Biology Review. Vol. 69, No. 3, p. 373-392, 2005). There are various techniques which can be applied to generate mutations in a genic sequence, and of particular importance in the present invention is the DNA shuffling technique (Rosic, N. N., Huang, W., Johnston, W. A., James J. Devoss, J. J., Gillam, E. M. J. Extending the diversity of cytochrome P450 enzymes by DNA family shuffling. Gene, Vol. 35762, No of Pages 9, 2007; Ling Yuan, L. Kurek, I., English, J. and Keenan, R. Laboratory-directed protein evolution. Microbiology and Molecular Biology Reviews, Vol. 69, No. 3, p. 373-392, 2005; Abécassis, V., Pompon, D. and Truan, G. High efficiency family shuffling based on multistep PCR and in vivo DNA recombination in yeast: statistical analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Research, Vol. 28, No. 20: E 88, 2000; Zhao, H. and Arnold, F. H. Functional and nonfunctional mutations distinguished by random recombination of homologous genes. Proc. Natl. Acad. Sci. U.S.A., Vol. 94, p. 7997-8000, 1997; Stemmer, W. P. C. Rapid evolution of a protein in vitro By DNA shuffling. Nature. London, Vol. 370, p. 389-391, 1994).
The technique of DNA shuffling consists of a directed molecular evolution, which generates punctual changes in the primary structure of the DNA molecules by means of random mutations (Ling Yuan, L. Kurek, I., English, J. and Keenan, R. Laboratory-directed protein evolution. Microbiology and Molecular Biology Reviews, Vol. 69, No. 3, p. 373-392, 2005; Stemmer, W. P. C. Rapid evolution of a protein in vitro By DNA shuffling. Nature. London, Vol. 370, p. 389-391, 1994, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721). Firstly, the gene of interest is randomly fragmented into small sequences of 30-50 base pairs, and this product is recombined in a PCR reaction (Polymerase Chain Reaction), which is conducted without the addition of oligonucleotides. In a second consecutive reaction, the products of the first reaction and specific oligonucleotides are added. Thus, a population of analog mutant/variant genes can be amplified (Stemmer, W. P. C. Rapid evolution of a protein in vitro by DNA shuffling. Nature. London, Vol. 370, p. 389-391, 1994; Zhao, H. and Arnold, F. H. Functional and nonfunctional mutations distinguished by random recombination of homologous genes. Proc. Natl. Acad. Sci. U.S.A., Vol. 94, p. 7997-8000, 1997).
The efficiency of the technique in producing analog molecules with greater biological activity has been proven in various works such as, for example, in Jager et al (Jager, S. A. W., Jekel, P. A. and Janssen, D. B. Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics. Enzyme And Microbial Technology, Vol. 40, p. 1335-1344, 2007), where the enzyme activity of the penicillin acyclase increased by 90%. The technique can use a single gene or more homologous genes and its success depends on a delicate arrangement between the size of the library, the biological diversity of origin, and a selection methodology of the variants having the desired characteristic (Ling Yuan, L. Kurek, I., English, J. and Keenan, R. Laboratory-directed protein evolution. Microbiology and Molecular Biology Reviews, Vol. 69, No. 3, p. 373-392, 2005).
The association of DNA shuffling techniques (creation of combinatorial libraries) and presentation of proteins on the surface of bacteriophages—Phage Display, makes selecting and expressing new molecules much more efficient (Stoop, A. A., Jespers, L., Lasters, I., Eldering, E. And Pannekoek, H. High-density mutagenesis by combined DNA shuffling and phage display to assign essential amino acid residues in protein-protein interactions: application to study structure-function of plasminogen activation inhibitor 1 (PAI-I). J. Mol. Biol., Vol. 301, p. 1135-1147, 2000).